Articular cartilage of the knee is quite susceptible to injuries which are often painful and which may progress to osteoarthritis. Osteochondral autografting and allografting are currently the only treatment options which immediately restore hyaline cartilage to the injured joint. Drawbacks to these procedures such as donor site morbidity and limited tissue availability have stimulated interest in osteochondral xenografts, which have the potential advantages of low cost and abundant supply. The long-term objective of the proposed research is to develop a decellularized porcine osteochondral xenograft (OCXG) which can be available for implantation as soon as a cartilage lesion is discovered (i.e., off-the-shelf). Development includes optimization of decellularization and crosslinking methods to reduce immunogenicity and improve graft durability and integration with host tissue. The specific Aims are as follows: 1. Compare glycosaminoglycan-containing and glycosaminoglycan-free porcine osteochondral dowels in terms of their mechanical properties and ability to support stem cell infiltration and chondroinduction in vitro. 2. Evaluate the effect of four different plant- derived, nontoxic crosslinking agents on the physical and mechanical properties of GAG(+) and GAG(-) OCXGs, as well as their effects on primary human knee chondrocyte behavior in vitro. 3. Evaluate the acute inflammatory response to GAG(+) and GAG(-) decellularized porcine OCXGs, both crosslinked and non- crosslinked using the most promising agent from SA2, in an intraarticular rabbit model. 4. Determine the capacity for crosslinked, decellularized OCXGs to repair full-thickness defects in the trochlear groove of adult dogs. The first aim will provide important information about fundamentally different approaches to decellularization, one which seeks to preserve all extracellular matrix and one which intentionally extracts non- collagenous components (especially glycosaminoglycan) to create more porosity. Evaluations include measurement of the DNA and GAG contents, the graft cartilage's biphasic properties, and the attachment, proliferation, and matrix production of human chondrocytes cultured on the grafts. The first aim also includes development of a novel decellularization protocol. The second aim will reveal the potential for natural, nontoxic crosslinking agents to enhance xenograft mechanical properties and collagenase resistance. It will also determine whether chondrocytes are sensitive to the degree of crosslinking. The third aim involves short-term transplantation of OCXGs into the medial femoral condyle of mature rabbits, mainly to investigate OCXG- induced synovitis. The effects of decellularization method and crosslinking will be examined through synovial fluid analysis and histology. The final aim will evaluate the functionality of porcine osteochondral xenografts used to fill surgically created defects in the canine knee joint. Semiquantitative scoring of macroscopic appearance and histology, in addition to indentation testing, will demonstrate whether osteochondral xengrafts are capable of restoring a healthy joint surface in this preclinical model.